Multidrug-resistant Lung Cancer Research Articles

MiRNA 146a promotes chemotherapy resistance in lung cancer cells by targeting DNA damage inducible transcript 3 (CHOP)

The underlying molecular mechanism of lung cancer drug resistance is poorly understood. The mediator of endoplasmic reticulum stress CHOP (DNA damage inducible transcript 3) promotes stress-induced apoptosis and appears to function as a transcription factor in multiple diseases. However, its potential contributions to multidrug resistance in solid tumors is unknown. Here, we investigated CHOP expression in tumor tissues form 69 lung cancer patients, finding that deficient CHOP expression is associated with poor prognosis. Cisplatin-resistant lung cancer cells exhibited lower expression of CHOP compared to that in sensitive lung cancer cells, and silencing or augmenting CHOP expression enhanced or impaired cisplatin resistance, respectively. Mechanistic investigations revealed that CHOP is directly associated with the regulation of autophagy or apoptosis-regulatory genes including LC3-II, death receptor 5 (DR5), and telomere repeat-binding factor 3. Notably, CHOP was identified as a target of miR-146a, and increased miR-146a expression in lung cancer cells was suggested to be responsible for CHOP mRNA down-regulation. Further, animal models confirmed that abnormally expressed miR-146a in lung cancer cells does not affect growth, but rather alters chemotherapy sensitivity. Together, CHOP is a useful prognostic marker and miR-146a is a potential therapeutic target for multidrug-resistant lung cancer.

Synthesis and biological evaluation of certain hydrazonoindolin-2-one derivatives as new potent anti-proliferative agents

In connection with our research program on the development of novel indolin-2-one-based anticancer candidates, herein we report the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l. The synthesised derivatives were in vitro evaluated for their anti-proliferative activity towards lung A-549, colon HT-29, and breast ZR-75 human cancer cell lines. Compounds 5b, 5c, 7b, and 10e emerged as the most potent derivatives with average IC50 values of 4.37, 2.53, 2.14, and 4.66 µM, respectively, which are superior to Sunitinib (average IC50 = 8.11 µM). Furthermore, compounds 7b and 10e were evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 cancer cell line. Moreover, 7b and 10e inhibited the cell growth of the multidrug-resistant lung cancer NCI-H69AR cell line with IC50 = 16 µM. In addition, the cytotoxic activities of 7b and 10e were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss-3t3) where both compounds displayed mean tumor selectivity index (1.6 and 1.8) higher than that of Sunitinib (1.4).

Role of TGF-β1 in multi-drug resistance in small cell lung cancer and its clinical significance

To investigate the role of transforming growth factor β1 (TGF-β1) in multi-drug resistance in small cell lung cancer and its clinical significance. Methods: The mRNA and protein expressions of TGF-β1 in H69 and H69AR cells were detected by real-time PCR and Western blot, respectively. After silence of TGF-β1, the sensitivity of H69AR to drugs was detected by CCK8 assay. The expressions of TGF-β1 in lung cancer and paracarcinoma tissues were examined by QRT-PCR and immunohistochemistry. The relationship of TGF-β1 expression with clinical pathological features and prognosis of patients was studied. Results: Compared to H69, the mRNA and protein expressions of TGF-β1 in H69AR cells were significantly increased by (5.93±0.47) and (8.49±1.92) folds, respectively (P<0.01). Transfection of TGF-β1 siRNA resulted in a decrease of TGF-β1 expression by 70.432% in H69AR cells (F=21.20, P<0.01) and an increase insensitivity to chemotherapeutic agents of H69AR cells (t=4.576, P<0.05). Compare with the paracarcinoma tissues, the expression of TGF-β1 was significantly increased in small cell lung cancer tissues (t=13.925, P<0.01), which was closely related with clinical stage, chemosensitivity and overall survival (all P<0.05), but not related with gender, age (both P>0.05). Conclusion: TGF-β1 is involved in the regulation of small cell lung cancer multidrug resistance, which may be a potential marker to evaluate the chemosensitivity and clinical prognostic for small cell lung cancer.

Identification of multidrug resistance gene MDR1 associated microRNA of salvianolic acid A reversal in lung cancer

This paper was aimed to investigate the microRNA associated with multidrug resistance gene MDR1 of salvianolic acid A reversal in lung cance. Human lung cancer A549 cells were divided into normal control group and drug group, and the MDR1 expression levels were determined by real-time quantitative PCR. MicroRNA expression profiling of normal control group and drug group were detected by using the latest microRNA microarray. Quantitative RT-PCR was used to validate the differentially expressed miRNA. Forecast of miRNA associated with MDR1 multi-resistant genes of up-regulated miRNA. Experimental results showed that the dosage of MDR1 expression level significantly lowered compared with control group. The miRNA expression spectrum analyses of human lung cancer A549 cells to drug group and the control group were detected by microRNA microarray, 426 differentially expressed miRNA were screened out. Then target prediction were performed for difference up-expression of miRNA and found that there were four obvious increase of miRNA associated with MDR1 multi-resistant genes. Real-time quantitative PCR for 4 microRNA verification, the results were consistent with the chip. So the author considered that salvianolic acid A down lung cancer multidrug resistance gene MDR1 is likely to be affected by the miRNA expression and regulation of target genes, to further clarify the traditional Chinese medicine to reverse multi-drug resistant mechanism provides the experimental basis.

Upregulation of ABCG2 via the PI3K-Akt pathway contributes to acidic microenvironment-induced cisplatin resistance in A549 and LTEP-a-2 lung cancer cells.

Hypoxia always exists in the processes involved in the development of lung cancer and contributes to an acidic microenvironment. Despite that hypoxia in the tumor microenvironment is associated with the formation of chemotherapeutic resistance, the exact role of an acidic microenvironment in the development of hypoxia-induced lung cancer multidrug resistance is still unknown. In the present study, we acidized the medium with 2-(N-morpholino)-ethanesulfonic acid (MES monohydrate) to mimic the acidic tumor microenvironment and observed the effects of acidification on lung cancer cell viability, the expression of ATP-binding cassette sub-familyG member2 (ABCG2) and myeloid cell leukemia‑1 (Mcl-1), and activation of the PI3K-Akt pathway. Thereafter, we investigated the mechanisms involved in the acidification-induced expression of ABCG2 and Mcl-1, and the potential therapeutic strategy to overcome acidification-associated multidrug resistance formation. We demonstrated that acidification obviously increased the expression of ABCG2 and Mcl-1 via PI3K‑Akt‑mTOR-S6 pathway activation and contributed to multidrug resistance. Inhibition of PI3K-Akt activity efficiently abolished the effect of acidification on cell viability, indicating that the PI3K-Akt pathway may include potential therapeutic target molecules in acidized microenvironment-associated lung cancer chemotherapeutic resistance.

The effect and mechanism of microRNA-21 on cis-dichlorodiamineplatinum resistance in lung cancer cell strain

To investigate the role of miR-21 on multidrug resistance (MDR) in non-small cell lung cancer(NSCLC)and to provide experimental and theoretical basis for MDR reversal. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to determine the mRNA level of miR-21 both in the chemo-sensitivity cell strain A549 and the chemo-resistance cell strain A549/DDP (primary A549/DDP cells). The miR-21 interference sequence was synthesized and transfected into A549/DDP cells by liposome as the carrier. The miR-21 expression level was knocked down and the change of chemo-sensitivity of cells was detected. Effects of miR-21 on the cell apoptosis and cell cycle in miR-21-depleted A549/DDP cells were analyzed by flow cytometry to elucidate the involvement of miR-21 in MDR reversal in NSCLC. The expression of multidrug-resistant proteins Survivin, Cyclin D1, Epidermal growth factor receptor (EGFR), Multidrug resistance-associated protein1 (MRP1) and Lipoprotein receptor-related protein (LRP) were detected by Western blot. MiR-21 level in multidrug-resistant NSCLC cell line A549/DDP was (5.223±0.316) folds higher than that in A549 cell line (t=48.318, P<0.01). Knockdown of miR-21 in A549/DDP cells significantly reversed their sensitivity to cis-DDP, meanwhile, the value of half maximal inhibitory concentration was significantly decreased compared with control group transfected with empty vector ((22.2±1.2) and (48.6±3.2) μmol/L, t=5.608, P<0.01). Cell apoptosis rate in A549/DDP cell with knockdown of miR-21 was significantly increased compared with primary A549/DDP cell line ((27.7±1.1) % and (16.8±1.1) %, t=10.183, P<0.01). Cell cycle G0/G1 phase in A549/DDP cell with knockdown of miR-21 was significantly decreased compared with primary A549/DDP cell line ((37.5±1.2) % and (43.4±2.3) %, t=8.202, P<0.01). Compared with primary A549/DDP cell line, the expression of Survivin, Cyclin D1, EGFR, MRP1 and LRP in A549/DDP cell with knockdown of miR-21 were significantly decreased ((71.7±4.3)%, t=8.325, P<0.01; (69.4±4.5)%, t=5.162, P<0.01; (52.3±3.2)%, t=8.042, P<0.01; (60.6±4.2)%, t=6.641, P<0.01; (72.9±3.8)%, t=4.566, P<0.01, respectively). miR-21 silencing reverses the multidrug resistance in lung cancer cell via accelerating cell apoptosis and modulating multidrug resistance-related gene expression. miR-21 may be a potential therapeutic candidate in multidrug resistance patients with lung cancer.

Abstract LB-292: Epigenetic downregulation of ABCG2 in colorectal cancer by a novel microRNA-DNA methylation mechanism - an attractive target for cancer prevention

Abstract Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. It develops in a complex multi-step process. The majority of studies to date have focused on genetic mutations and epigenetic changes that drive the CRC carcinogenesis process. Xenobiotic transporters play an important role in safeguarding our body from external toxic substances. These transporters lining the gastrointestinal tract may thus protect us from the dietary carcinogens. This study aims to investigate the downregulation of an efflux transporter ABCG2 in colorectal cancer versus normal colon mucosa, so as to shed some light on its relevance to CRC initiation and progression. We found that ABCG2 expression is at least 50-fold lower in adenomatous polyps and colon carcinoma specimens obtained from CRC patients than in their matched pair of adjacent normal colon mucosa. This may facilitate the accumulation of genotoxins to propel tumor initiation and progression. The underlying mechanism(s) for ABCG2 under-expression in CRC is currently not known. To this end, aberrant promoter methylation of ABCG2 has been reported to cause its repression in a few cancer types including renal carcinoma and multiple myeloma. On the other hand, ABCG2 promoter demethylation has been reported to upregulate the transporter to mediate multidrug resistance in lung cancer and acute lymphoblastic leukemia. In this study, miR-203 was found to be downregulated in all polyps and CRC specimens, relative to adjacent normal colon mucosa. We demonstrated that the de novo DNA methyltransferase DNMT3 is a direct target of miR-203. Importantly, by relieving the repression on DNMT3, the lower expression of miR-203 in CRC caused remarkable ABCG2 promoter methylation and lower ABCG2 expression in colon cancer cell lines and the patient CRC specimens. In spite of the complex sequential genetic mutations and epigenetic changes leading to CRC formation, we proposed that the restoration of the xenobiotic transporter ABCG2 function via modulating this new microRNA-DNA methylation mechanism in precancerous cells may represent an attractive strategy to stop or delay the carcinogenesis process. Citation Format: Kenneth K.W. To, Wing Wah Leung, Simon S.M. Ng. Epigenetic downregulation of ABCG2 in colorectal cancer by a novel microRNA-DNA methylation mechanism - an attractive target for cancer prevention. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-292. doi:10.1158/1538-7445.AM2015-LB-292

Multidrug resistance characterization in multicellular tumour spheroids from two human lung cancer cell lines.

BackgroundMost of the knowledge about the mechanisms of multidrug resistance in lung cancer has been achieved through the use of cell lines isolated from tumours cultivated either in suspensions of isolated cells or in monolayers and following exposition to different cytostatic agents. However, tumour cell lines growing as multicellular tumour spheroids (MTS) frequently develop multicellular resistance in a drug-independent form. The aim of this study was to characterize the phenotypic and functional differences between two human NSCLC cell lines (INER-37 and INER-51) grown as traditional monolayer cultures versus as MTS.MethodsAfter 72 hours treatment with anticancer drugs, chemosensitivity in monolayers and tumour spheroids cultures was assessed using MTT assay. Reverse transcription-polymerase chain reaction was employed to detect the mRNAs of multidrug resistance-related genes. The expression of P-gp was analyzed by immunohistochemical staining and cell cycle profiles were analyzed using FACS.ResultsThe results indicate that when grown as MTS each lung cancer cell line had different morphologies as well as and abrogation of cell proliferation with decrease of the G2/M phase. Also, MTS acquired multicellular resistance to several chemotherapeutic agents in only a few days of culture which were accomplished by significant changes in the expression of MDR-related genes.ConclusionOverall, the MTS culture changed the cellular response to drugs nevertheless each of the cell lines studied seems to implement different mechanisms to acquire multicellular resistance.

Role of SPHK1 regulates multi-drug resistance of small cell lung cancer and its clinical significance

背景与目的小细胞肺癌约占全部肺癌的15%，化疗是其主要的治疗方法之一，虽然早期对一线化疗方案敏感，但极易出现多药耐药而导致治疗失败。前期基因芯片发现SPHK1与小细胞肺癌的耐药性相关，本研究进一步探讨SPHK1在小细胞肺癌多药耐药中的作用。方法首先通过QRT-PCR和Western blot从基因和蛋白水平检测化疗敏感细胞株H69及多药耐药细胞株H69AR中SPHK1的差异表达；转染siRNA下调H69AR细胞中的SPHK1的表达，通过CCK8检测细胞对各种化疗药物（ADM, DDP, VP-16）的敏感性变化，流式细胞仪检测细胞周期及凋亡的变化。同时收集小细胞肺癌化疗前组织和血液标本，将其分为化疗敏感组和耐药组，QRT-PCR检测小细胞肺癌患者血液标本中SPHK1的表达，免疫组化法检测小细胞肺癌患者组织标本中SPHK1的表达，分析SPHK1与小细胞肺癌患者预后相关性。结果SPHK1在耐药细胞H69AR中的表达明显高于H69，下调H69AR中SPHK1的表达能够增加细胞对化疗药物的敏感性，促进细胞的凋亡，细胞周期发生G0/G1期阻滞，SPHK1在小细胞肺癌耐药患者中的表达较敏感患者明显增加，SPHK1的表达与患者的性别、年龄无关，与疾病的分期、对化疗的敏感性及生存时间密切相关，差异具有统计学意义（P＜0.05）。结论SPHK1参与调节小细胞肺癌多药耐药，SPHK1可作为评估小细胞肺癌化疗敏感性及临床预后的潜在靶基因。

Effect and mechanism of Src tyrosine kinase inhibitor sunitinib on the drug-resistance reversal of human A549/DDP cisplatin-resistant lung cancer cell line.

The development of tumor cell drug resistance is the primary reason for treatment failure in lung cancer chemotherapy. Therefore, overcoming multidrug resistance is currently an urgent issue to be addressed in lung cancer treatment. Sunitinib is a tyrosine kinase inhibitor with confirmed inhibitory effects on tumor growth and metastasis; however, the effects of sunitinib and mechanisms of action in lung cancer multidrug resistance are yet to be determined. The present study was designed to examine the effects of sunitinib and the mechanisms underlying lung cancer multidrug resistance. It was observed that sunitinib was able to improve the sensitivity of A549/DDP lung cancer cells to cisplatin, enhance tumor apoptosis, arrest the cell cycle in G0/G1 phase, upregulate intracellular Rh-123 content, downregulate the expression of P-glycoprotein, multidrug resistance protein 1, multidrug resistance-associated protein 1, lung resistance protein, glutathione-S-transferase, ERCC1, survivin and Bcl-2 in tumor cells, phosphorylation of AKT and extracellular signal-regulated kinase (ERK), glutathione activity, and transcriptional activity of nuclear factor-κB, Twist, Snail and AP-1. The results demonstrated that sunitinib was able to reverse the multidrug resistance of A549/DDP lung cancer cells, which was possibly associated with the downregulation of multidrug resistance-associated gene expression and the inhibition of AKT and ERK phosphorylation.

Feroniellin A-induced autophagy causes apoptosis in multidrug-resistant human A549 lung cancer cells

During the screening of natural chemicals that can reverse multidrug resistance in human A549 lung cancer cells resistant to etoposide (A549RT-eto), we discovered that Feroniellin A (FERO), a novel furanocoumarin, shows toxicity toward A549RT-eto cells in a dose- and time-dependent manner. FERO reduced the expression of NF-κB, leading to downregulation of P-glycoprotein (P-gp), encoded by MDR1, which eventually sensitized A549RT-eto cells to apoptosis. FERO specifically diminished transcription and promoter activity of MDR1 but did not inhibit the expression of other multidrug resistance genes MRP2 and BCRP. Moreover, co-administration of FERO with Bay11-7802, an inhibitor of NF-κB, accelerated apoptosis of A549RT-eto cells through decreased expression of P-gp, indicating that NF-κB is involved in multidrug resistance. Conversely, addition of Z-VAD, a pan-caspase inhibitor, blocked FERO-induced apoptosis in A549RT-eto cells but did not block downregulation of P-gp, indicating that a decrease in P-gp expression is necessary but not sufficient for FERO-induced apoptosis. Interestingly, we found that FERO also induces autophagy, which is characterized by the conversion of LC3 I to LC3 II, induction of GFP-LC3 puncta, enhanced expression of Beclin-1 and ATG5, and inactivation of mTOR. Furthermore, suppression of Beclin-1 by siRNA reduced FERO-induced apoptosis in A549RT-eto cells and activation of autophagy by rapamycin accelerated FERO-induced apoptosis, suggesting that autophagy plays an active role in FERO-induced apoptosis. Herein, we report that FERO reverses multidrug resistance in A549RT-eto cells and exerts its cytotoxic effect by induction of both autophagy and apoptosis, which suggests that FERO can be a useful anticancer drug for multidrug-resistant lung cancer.

Nanoparticle Chemosensitization

Nanoparticle Chemosensitization

The novel toluidine sulphonamide EL102 shows pre-clinical in vitro and in vivo activity against prostate cancer and circumvents MDR1 resistance

Background:Taxanes are routinely used for the treatment of prostate cancer, however the majority of patients eventually develop resistance. We investigated the potential efficacy of EL102, a novel toluidine sulphonamide, in pre-clinical models of prostate cancer.Methods:The effect of EL102 and/or docetaxel on PC-3, DU145, 22Rv1 and CWR22 prostate cancer cells was assessed using cell viability, cell cycle analysis and PARP cleavage assays. Tubulin polymerisation and immunofluorescence assays were used to assess tubulin dynamics. CWR22 xenograft murine model was used to assess effects on tumour proliferation. Multidrug-resistant lung cancer DLKPA was used to assess EL102 in a MDR1-mediated drug resistance background.Results:EL102 has in vitro activity against prostate cancer, characterised by accumulation in G2/M, induction of apoptosis, inhibition of Hif1α, and inhibition of tubulin polymerisation and decreased microtubule stability. In vivo, a combination of EL102 and docetaxel exhibits superior tumour inhibition. The DLKP cell line and multidrug-resistant DLKPA variant (which exhibits 205 to 691-fold greater resistance to docetaxel, paclitaxel, vincristine and doxorubicin) are equally sensitive to EL102.Conclusion:EL102 shows potential as both a single agent and within combination regimens for the treatment of prostate cancer, particularly in the chemoresistance setting.

Reversal of Lung Cancer Multidrug Resistance by pH-Responsive Micelleplexes Mediating Co-Delivery of siRNA and Paclitaxel

The recent advances in RNA interference (RNAi) technology provided novel and promising solutions for human cancer treatment. In this study, the application of dual pH-responsive cationic micellar nanoparticles for small interfering RNA (siRNA) and paclitaxel (PTX) co-delivery to overcome cancer multidrug resistance (MDR) is reported. The in vitro siRNA transfection shows that siRNA-luciferase (Luc) loaded micelleplexes efficiently silences Luc expression in various carcinoma cell lines. The Luc knockdown ability of the micelleplexes can be enhanced by choloquine (CQ) co-incubation. However, is abolished by bafilomycin-A1 (Baf-A1) treatment. The micelleplexes are further exploited for co-delivery of siRNA-Bcl-2 and PTX to Bcl-2 overexpressing A549 lung cancer cells (A549-Bcl-2). The experimental results show that the micelleplexes could sensitize A549-Bcl-2 cells to PTX via down-regulation of anti-apoptosis gene of Bcl-2, suggesting that PDMA-b-PDPA micelleplexes are promising nanovectors for siRNA and anti-cancer drug co-delivery to overcome cancer MDR.

Abstract 4522: Nanoparticle chemosensitization.

Abstract Systemic drug toxicity and multi-drug resistance are the two major obstacles in the treatment of cancer. While multi-drug resistance can be easily overcome by the use of chemosensitizers, the process of chemosensitization itself has been shown to be highly toxic to the normal cells. This is because chemosensitizers not only sensitize the tumor tissue but also the normal tissue to the effect of the drug leading to a substantial increase in the systemic toxicity of the drug. On the other hand, nanoparticles (NPs) can decrease systemic toxicity by virtue of their property of preferential accumulation at the site of tumor, making them an ideal candidate for the delivery of the chemosensitizers. We developed a novel method to overcome both of the major obstacles in cancer treatment by synthesizing NP formulations of two potent chemosensitizers, wortmannin (Wtmn) and olaparib. Wtmn is a highly effective PI3 kinase and DNA-PK inhibitor while olaparib is highly potent Poly ADP ribose polymerase (PARP) inhibitor. NP Wtmn and NP olaparib were synthesized using a biodegradable core-shell NP platform. We studied the chemosensitization effects of NP Wtmn and NP olaparib in vitro using the lung cancer cell lines H23 and H460, and a multi-drug resistant lung cancer cell line H69AR followed by the drug treatment. We used different chemotherapeutic drugs such as etoposide, gemcitabine and docetaxel to demonstrate the effect of NP chemosensitizers. The biodistribution of the NPs was determined using the mouse xenograft model of H460. Normal tissue toxicity was evaluated by using NP chemosensitizers and compared with their small molecule counterparts. Finally, we evaluated NP Wtmn and NP olaparib as a chemosensitizers in vivo using murine xenograft models of H69AR and H460 and compared them to that of free chemosensitizers. NP chemosensitizers, similar to their small molecule counterparts, are highly potent in vitro in all three cell lines. The biodistribution study showed that NP formulation leads to a higher tumor accumulation when compared to the free molecules. Toxicity study showed significantly less hepatic and hematologic toxicity with NP formulation than the free molecules. In vivo efficacy study demonstrated that NP Wtmn and NP olaparib are potent chemosensitizers and are more effective than their small molecule counterparts. Further, we demonstrated that NP Wtmn can overcome drug resistance in H69AR cell line. Our work demonstrates the proof of principle in utilizing nanomedicine for the improving chemosensitization. Citation Format: Manish Sethi, Sonya R. Kowalczyk, Chintan H. Kapadia, Michael E. Werner, Rohit Sukumar, Edina Wang, Andrew Z. Wang. Nanoparticle chemosensitization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4522. doi:10.1158/1538-7445.AM2013-4522

Enhanced antitumor efficacy by Paclitaxel-loaded Pluronic P123/F127 mixed micelles against non-small cell lung cancer based on passive tumor targeting and modulation of drug resistance

The aim of this work was to demonstrate the advantage of using paclitaxel (PTX)-loaded Pluronic P123/F127 mixed micelles (PF-PTX) against non-small cell lung cancer (NSCLC) compared to Taxol. Modulation of multidrug resistance (MDR) by Pluronic mixed micelles was evaluated in lung resistance protein (LRP)-overexpressing human lung adenocarcinoma A-549 cell line. Influence of PF-PTX on in vitro cytotoxicity was determined by MTT assay, while cellular apoptosis was detected by cell nuclei staining and Annexin V-FITC apoptosis detection kit. Cell cycle arrest was also confirmed by flow cytometry. Additionally, in vivo fate and antitumor efficacy of PF-PTX were extensively evaluated in comparison with Taxol. It was demonstrated that PF-PTX had superior anti-proliferation activity against A-549 cells compared with Taxol as measured by IC 50. The enhanced anti-cancer efficacy of PF-PTX was associated with PTX-induced apoptosis and cell arrest in the G 2/M phase. Intracellular ATP depletion and decreased mitochondrial potential caused by Pluronic copolymers were found to be related to modulation of MDR. PF-PTX also exhibited significant advantages in pharmacokinetics and A-549 xenograft tumor model versus Taxol. The PF-PTX formulation achieved 3.0-fold longer mean residence time in circulation, 2.2-fold larger area under the plasma concentration–time curve than Taxol. At 28 days, tumor volume in PF-PTX group was only 31.8% that of the Taxol. Therefore, PF-PTX significantly enhanced the anti-cancer activity of PTX and might be considered a promising drug delivery system to overcome MDR in lung cancer.

Abstract LB-283: Novel small molecule has potent antitumor activity in drug-resistant human carcinoma xenograft models

Abstract We have previously shown that Kevetrin, a novel molecule currently under development, was effective in reducing tumor growth in human multi-drug resistant lung cancer xenograft models while being well tolerated. In this study, we expanded our tests to include other tumor types: colon and breast. Three of the models have been shown to be multi-drug resistant: HCT-15 colon carcinoma have an overexpression of p-glycoprotein that acts as a membrane efflux pump (Iwahashi 1991, Mickley 1989), MDA-MB-435s carcinoma cells have highly active c-Abl and Arg kinases and overexpress Heat Shock Protein 27 which inhibits apoptosis thereby contributing to drug resistance (2006 Srinivasan, 2008 Shi), and A549 lung carcinoma have the K-ras mutation and overexpress STAT3 and Nrf2; mutations associated with resistance to standard chemotherapy (Akudela 2004, Mahaffey 2009, Homma 2009, Kim 2009). To demonstrate a dose response effect, in the multi-drug resistant A549 lung tumor model, nude mice bearing established tumors were treated with either 50, 100, or 200 mg/kg Kevetrin intraperitoneally (IP) every other day for 3 doses or 200 mg/kg every other day for 3 doses followed by a second cycle 10 days later. Kevetrin delayed median tumor growth in a dose dependent manner where 50, 100, and 200 mg/kg produced tumor growth delays (TGDs) of 0%, 6%, and 21%, respectively, relative to controls. When given in 2 cycles at 200 mg/kg, a significantly greater TGD of 35% was achieved. In the HCT-15 multi-drug resistant colon tumor model, nude mice bearing established tumors were treated with either 200 mg/kg Kevetrin IP every other day for 3 doses or 22 mg/kg paclitaxel intravenously (IV) daily for 4 doses. Paclitaxel had no anti-tumor activity, whereas Kevetrin produced a significant TGD of 16 days (43%) relative to controls. In a non drug-resistant colon tumor model, HT-29, Kevetrin produced a similar TGD of 18 days (49%) while 20 mg/kg 5-fluorouracil (5-FU) IP given daily for 5 doses produced a TGD of 9 days (24%). In the MDA-MB-435s drug resistant tumor model, nude mice bearing established tumors were treated as above. Paclitaxel had no significant anti-tumor activity (1% TGD), whereas Kevetrin produced a significant TGD of 20 days (65%). In a non drug-resistant breast tumor model, MDA-MB-231, Kevetrin produced a TGD of 26 days (90%), and paclitaxel produced a significant TGD of 18 days (62%). These results demonstrate that Kevetrin exerted potent anti-tumor activity against three different tumor types, e.g., lung, breast, colon, including those that have multi-drug resistant phenotypes at doses and schedules that were well-tolerated as suggested by &amp;lt;5% weight loss during treatments. Therefore, these studies suggest that Kevetrin has promising potential for the treatment of tumors that have become resistant to standard chemotherapy. Based on our studies we plan to initiate a Phase I clinical trial in solid tumors in 2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-283.

The Human Lung Cancer Drug Resistance-Related Gene BC006151 Regulates Chemosensitivity in H446/CDDP Cells

This study aimed to investigate the mechanism by which the human lung cancer drug resistance-related gene BC006151 regulates chemosensitivity by down-regulating BC006151 expression via antisense gene transfer in H446/(C)DDP cells. A retroviral vector containing the antisense BC006151 sequence was constructed and transfected into H446/(C)DDP cells. Transfection of the empty vector served as a negative control. The two groups of transfected cells were treated with various chemotherapeutic agents, after which morphological changes in cell ultrastructure were compared by transmission electron microscopy, cell proliferation and chemosensitivity to particular chemotherapeutic agents were compared by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, the effects of chemotherapy on cell cycle and apoptosis were compared by flow cytometry, and Bcl-2 was evaluated by immunohistochemistry and Western blot analysis. Results showed that apoptotic body-like structures were identified by transmission electron microscopy in the antisense gene-transfected cells. MTT founded that these cells exhibited a significantly lower level of proliferation than the control cells (p<0.01), together with a markedly increased sensitivity to various chemotherapeutic agents (p<0.01). Flow cytometry analysis revealed that a G1 phase arrest accounted for the reduction in proliferation in the antisense gene-transfected cells; increased apoptosis was also detected (p<0.01). Both immunohistochemistry and western blot analysis confirmed that Bcl-2 expression was significantly down-regulated in the antisense gene-transfected cells compared to controls. In a word, down-regulation of BC006151 can significantly inhibit proliferation and increase apoptosis of H446/(C)DDP cells after chemotherapy, and this gene may play an important role in the development of multidrug resistance in lung cancer.

Influence of SiRNA Targeting Survivin on Chemosensitivity of H460/cDDP Lung Cancer Cells

We investigated the difference in survivin expression between a multidrug-resistant lung cancer cell line (H460/cDDP) and its parental counterpart (H460) and the influence of siRNA targeting survivin on the chemosensitivity of H460/cDDP. SiRNA targeting survivin was transfected into H460/cDDP cells using a liposome approach. Survivin mRNA and protein expression were significantly higher in H460/cDDP than H460 cells. The median inhibitory concentrations (IC(50)s) for cisplatin and paclitaxol in vitro against H460/cDDP cells were significantly lower in cells treated with survivin-specific siRNA than in control cells. Apoptosis and cleaved caspase-3 expression were analysed using annexin V and Western blotting, respectively, and showed a significant increase in apoptosis after treatment with the chemotherapeutic agents plus specific siRNA. Specific siRNA sensitized H460/cDDP cells to both cisplatin and paclitaxol. Thus, survivin appears to participate in the multidrug resistance mechanism of H460/cDDP cells and siRNA targeting survivin has the potential to increase the sensitivity of drug-resistant cancer cells to anticancer drugs.

The MDR1/ABCB1 regional amplification in large inverted repeats with asymmetric sequences and microhomologies at the junction sites

A multidrug-resistant lung cancer cell line PTX250, established by treatment with the anti-cancer drug paclitaxel, has been demonstrated to have an increased copy number in the 7q21.12 region including the MDR1/ABCB1 gene. The amplicon is 2.7 megabases in size, and the copy number increase is 11-fold compared with the parental cell line. Here, we examined the amplicon structure and determined nucleotide sequences at both junctions of the amplicon. Fluorescence in situ hybridization analysis using an MDR1 probe demonstrated a cluster of fluorescent signals at the chromosomal end, suggesting an intra-chromosomal amplification. DNA fragments of both junctions were cloned and sequenced. The distal junction was a head-to-head fusion with a 4-base pair (bp) overlap separated by an asymmetric sequence of 1,265 bp, and the proximal junction was a tail-to-tail fusion with a 2-bp overlap intervened by an asymmetric sequence of 2,203 bp. These results suggest that the amplicon has a large palindromic structure with an asymmetric sequence and has been amplified through the breakage-fusion-bridge cycle. Specific sequences, which might be related to the occurrence of double-strand-breakages, were found at or near the junctions of the amplicon — an inverted repeat in the distal junction and a highly AT-rich region near the proximal junction.